Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol

Abstract

A new anticalin against estradiol (E₂), a kind of endocrine disruptor, was obtained in the present study to detect E₂ levels. A member of the lipocalin family from Pieris brassicae called bilin-binding protein (BBP) was employed for the preparation of a random library to specifically complex E₂. Sixteen amino acid residues at the center of the binding site, which were formed by four loops on top of an eight-stranded β-barrel, were subjected to targeted random mutagenesis. Estradiol-binding BBP variants so-called ‘anticalins’, which exhibit binding activity for compounds, such as E₂, were selected from the resulting library by combining both ribosome display and screening techniques. Four variants of complex E₂ with high affinity were identified. These variants exhibited dissociation constants (KDs) as low as 54.265 nM. ELISA showed that ribosome displayed anticalin (E₂–A) specifically bound E₂. The 50% inhibition concentration (IC₅₀) for E₂ was 50 ng mL⁻¹ and the limit of detection (LOD:IC₁₀) was 0.071 ng mL⁻¹. The experimental results suggest that E₂–A can be used as a potential anticalin to detect E₂ in animals.